Progressive loss in vitro immune response with tumor growth.

Peripheral blood lymphocytes from rats carrying a transplantable hepatoma were cultured in the presence of phytohemagglutinin (PHA), concanavalin A (ConA) or dextran sulfate (DS) at various times after tumor cell inoculation or after its surgical removal. Mitogen-induced lymphocyte transformation, measured by tritiated thymidine incorporation, declined as the tumor size increased, especially when cells were cultured in autologous serum. The response to PHA and ConA declined prior to the response to DS. This inhibition could not be removed by extensive washing of the cells, alteration of serum concentration, time of incubation or mitogen dose. Culture for 24 hr prior to the addition of high doses of mitogen resulted in partial restoration of the PHA and ConA, but not DS, responses. Previously inhibited responses also returned when the tumor was surgically removed. Spleen cells from animals with large tumors were also inhibited.     

Stimulation of chicken lymphocytes by T- and B-cell mitogens.

Cultures of chicken spleen, peripheral blood, thymus, and bursal lymphocytes were tested for mitogenic stimulation by phytohaemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), trypsin, and insulin. Spleen and blood leukocytes were stimulated by both the lectins and LPS, and also to some degree by trypsin and insulin as judged by increased incorporation of [³H]thymidine into acid-insoluble material. This was observed in cultures incubated in serum-free medium as well as in the presence of foetal bovine serum or autologous plasma. Thymus cells were reproducibly stimulated by high concentrations of PHA. No significant responses were obtained in bursal cell cultures with any of the compounds tested. Removal of cotton wool-adherent cells from the spleen cell suspensions resulted in a subpopulation of cells which were stimulated by PHA but showed little response to ConA, PWM, or LPS. This procedure did not remove surface immunoglobulin-bearing cells from the original suspension. Both these enriched spleen lymphocytes and the unfractionated spleen, blood and thymus leukocyte cultures were effectively stimulated by a partially purified PHA but with a highly purified PHA preparation only at very high concentrations. These and other results suggest that the mitogenic components in crude PHA preparations are different for chicken and human or mouse cells.     

Studies on rabbit lymphocytes in vitro : XX. Demonstration of cells reactive to more than one mitogen by mixed sequential stimulation

Rabbit peripheral blood lymphocyte (PBL) cultures stimulated by ConA and then blocked by the addition of competing sugar or antiserum after 6–15 h of ConA prestimulation respond to restimulation by PHA or PWM to a much greater extent than to continuous stimulation or delayed stimulation with PHA or PWM. This effect of mixed lectin sequential stimulation indicates that many of the same PBLs will respond to more than one mitogen, but that some cells require preactivation by one mitogen in order to respond fully to another mitogen. Thus, the number of PBLs which respond to PHA or PWM alone is much less than the number which respond following pretreatment with ConA when the pretreatment effect of ConA alone is blocked. Rabbit PBLs do not respond to LPS and preactivation by ConA does not prepare rabbit PBLs to respond to LPS.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Decrease in mitogen responsiveness of mononuclear cells from peripheral blood after epinephrine administration in humans

A single subcutaneous injection of 0.2 mg epinephrine into healthy human subjects caused a transient lymphocytosis in peripheral blood. Mononuclear cells (MNC), isolated at various times after epinephrine administration, were cultured in the presence of mitogens. The blastogenic responses to pokeweed mitogen (PWM) and phytohemagglutinin (PHA) were significantly reduced for up to 60 min post-epinephrine (p less than 0.05); the response to concanavalin A (Con A) was reduced in the 15-min samples only. All responses returned to pre-injection levels by 120 min post-injection. Removal of adherent monocytes from MNC isolates before culture did not restore normal mitogen responsiveness. When MNC were cultured in the absence of mitogens, there was no difference in survival between pre- and post-epinephrine samples. Incubation of untreated MNC for 2 hr or 18 hr in vitro with various concentrations of epinephrine (10(-5) to 10(-1) mg/ml) had no effect upon the subsequent blastogenic response to mitogens. Other workers have reported that epinephrine administration causes alterations in the composition of the circulating lymphocyte pool. Taken together, these data suggest that the reduction in mitogen responsiveness after epinephrine is the result of changes in the distribution of lymphocyte subclasses in peripheral blood.     

Modulation of phytohemagglutinin-mediated lymphocyte stimulation by egg lecithin

Egg lecithin at 200 μg/ml or greater was found to abrogate blastogenic responses of human leukocyte cultures stimulated with phytohemagglutinin (PHA), concanavalin A (ConA), purified protein derivatives of tuberculin (PPD), candidin or streptokinase and streptodornase (SKSD) while leukocyte aggregation mediated by PHA, however, occurred irrespective of the presence of the lipid. Inhibition of PHA-mediated responses occurred when the lecithin was added simultaneously with the mitogen but the extent of inhibition decreased with delay in the addition of the lipid. When added 48 h after their exposure to PHA, the presence of lecithin did not change the pattern of [³H]TdR incorporation. Prior sonic treatment of an aqueous suspension of lecithin was found to render the lipid more effective in arresting lymphocyte responses than the unsonicated control. In entity, these results indicated that the lipid did not affect the viability of the leukocyte cultures nor did it seem that the lipid may interfere with the binding of the mitogen per se. It was concluded therefore, that inhibition by lecithin may most likely occur early at a time following binding of the mitogen but before the cells are committed to cellular division and it seems that the plasma membrane might be a likely site of action of the lipid.     

Neuroimmunomodulation of the immune response by the endocrine system: effect of luteinizing hormone (LH) on the proliferative response of lymphocytes

Proliferative response of splenic lymphocytes from adult (2 months old) male mice to human luteotrophic hormone (hLH) was studied in vitro. hLH induced lymphoblastic transformation at a concentration of 0.1 micrograms/ml or higher. This hormone potentiated the mitogen activities of concanavalin A (ConA) and phytohemagglutinin (PHA) for T cells and of pokweed mitogen for B and T cells. The data suggest that peripherally circulating LH may regulate a certain function of lymphoid cells.     

Inhibition of ConA erythroagglutination by alkali-treated lipopolysaccharide

Bacterial lipopolysaccharide treated by hydrolysis in basic solution (hLPS) and then added to suspensions of human erythrocytes markedly inhibited erythroagglutination by concanavalin A (ConA), soybean agglutinin (SBA), and Phaseolus vulgaris phytohemagglutinin (PHA). Likewise, red cells presensitized by exposure to hLPS and then suspended in hLPS-free buffer were not agglutinated by ConA. However, when hLPS was not added to buffer both SBA and PHA strongly agglutinated erythrocytes presensitized by hLPS. Also, the binding of ³H-labeled ConA to red cells was unaffected by presensitization of the cells with hLPS. It appears, therefore, that membrane-associated hLPS localizes in the ConA receptor regions of erythrocyte membranes and inhibits ConA erythroagglutination by disruption of receptor responses to bound ConA or alteration of the active subunit structure of bound ConA.     

Separation of mitogen-induced suppressor cells of human antibody-producing cells

Human antibody-forming cells were demonstrated by a plaque in agar technique following in vitro stimulation with either pokeweed mitogen or Cowan I strain of protein A-positive Staphylococcus aureus bacteria. We evaluated the effects on this antibody formation caused by the addition of cells which had been stimulated with PH A or Con A. Both Con A and PHA cells harvested after 3 days showed strong inhibition of pokeweed-induced plaque formation. The majority of the suppression could be accounted for by a blast fraction separated on 1g sedimentation gradients from the Con A or PHA cultures. Small cells from such cultures showed inhibition of PFC when added at high ratios (1:2), but this suppressive activity diluted out much more rapidly than that of the blast cells. No helper activity was noted with either small cells or blasts. Our studies indicate a T-cell blast as the suppressive fraction in Con A- or PHA-stimulated human lymphoid cells. While this T-cell suppression applies to T-dependent responses such as antibody stimulation with pokeweed mitogen, it does not have a substantial effect on Cowan I-induced plaque-forming responses. The finding that Cowan I-induced plaques could not be inhibited by Con A or PHA blasts indicates the T independence of this response.     

A phorbol ester (TPA) can replace macrophages in human lymphocyte cultures stimulated with a mitogen but not with an antigen

A culture system was developed in which human peripheral blood mononuclear cells (PBM) depleted of macrophages did not proliferate in response to the lectin mitogen PHA or to the soluble antigen of tetanus toxoid. These cells were able to respond to both mitogen and antigen if purified autologous macrophages were added back to the culture. The response to PHA was partially restored by supplementing the cultures with supernatants from LPS-stimulated macrophages or with partially purified human interleukin 1 (IL 1). The response to tetanus was not restored by reconstitution with these materials. The phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), has been shown to have IL 1-like effects in other species and is a polyclonal activator of human T and B lymphocytes. In this study, we tested the ability of TPA to replace macrophages in human lymphocyte cultures stimulated with mitogen or with antigen. Small doses of TPA (50 ng/ml) completely replaced macrophages in the PHA-stimulated cultures; however, in doses of up to 400 ng/ml, TPA was not able to replace macrophages in cultures stimulated with tetanus. Thus, TPA appears to mimic the macrophage-replacing ability of soluble factors (IL 1, macrophage supernatants) in the triggering of human lymphocytes.     

Comparative study of mitogenic responses in man and Japanese monkeys (Macaca fuscata): responses of T-cell subsets, accessory cell dependency, and interleukin-2 receptor expression

Responses of peripheral blood mononuclear cells to phytohemagglutinin-P (PHA-P), concanavalin-A (ConA), and pokeweed mitogen (PWM) were compared in man and Japanese monkeys. Both CD8+ and CD8- T subsets showed greater responses to ConA than to PHA-P in the Japanese monkey. Addition of macrophages to each T subset produced more effective augmentation of ConA response in the Japanese monkey than in man, and ConA induced more interleukin-2-receptor-positive blast cells than PHA-P did in Japanese monkeys.     

Monocyte dependence of pokeweed mitogen-induced differentiation of immunoglobulin-secreting cells from human peripheral blood mononuclear cells.

Human peripheral blood mononuclear cells (PBM) lost the capacity to generate immunoglobulin-secreting cells (ISC) in response to pokeweed mitogen (PWM) when depleted of adherent cells (AC). The diminished responsiveness of the nonadherent cells (NAC) could not be ascribed to cell death, altered PWM dose response characteristics, or a change in the length of incubation required to generate a response. Supplementation with autologous or homologous AC, but not 2-mercaptoethanol, restored the capacity of NAC to generate ISC after PWM stimulation. By standard criteria AC were found to contain 85 to 90% monocytes. Furthermore, the monocytes and not the few lymphocytes contaminating the AC were responsible for restoring PWM responsiveness to the NAC. PWM-induced DNA synthesis of NAC also was markedly reduced compared to PBM. Again, supplementation with monocytes restored responsiveness to NAC. The monocyte dependence of PWM-induced proliferation and generation of ISC was most apparent when cultural conditions were employed that limited cell-to-cell interaction.     

Freezing of rat lymphocytes. I. The effects of dimethyl sulfoxide and freezing on the phytohemagglutinin- and pokeweed mitogen-responding lymphocyte subpopulations.

Rat spleen and lymph node lymphocytes have been frozen with dimethyl sulfoxide (DMSO) at 1 °C/min and stored at ?196 °C for 10 min. The functional recovery of the cell populations was monitored by the mitogenic response (uptake of [³H]thymidine) to phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in culture after thawing. With 5 to 10% DMSO in the freezing medium, frozen-thawed lymph node cells were found to retain about 40% of their response to PHA. In contrast, frozen-thawed spleen cells responded better to PHA than fresh cells. The response to PWM was markedly decreased in both spleen and lymph node cell cultures.A similar effect was observed when DMSO was added to the culture medium of fresh spleen cells, i.e., an augmentation of the response to PHA and a suppression of the response to PWM. However, the concentrations of DMSO needed to induce this effect was more than 10-fold higher than that present in the culture medium after freezing and thawing.Since removal of adherent cells from the spleen cell population also produced an augmentation of the response to PHA, it is suggested that the freezing procedure and DMSO may have an inhibitory effect on suppressor cell functions present in spleen cell populations.     

Possible mechanism of the preventive effect of BCG against diabetes mellitus in NOD mouse. I. Generation of suppressor macrophages in spleen cells of BCG-vaccinated mice.

With the aim of clarifying the mechanism of the suppressive action of BCG against insulitis and overt diabetes in NOD mice, we studied the effects of BCG on spleen cell populations and on the in vitro immune responses of spleen cells. The spleen cells of BCG-vaccinated mice showed much lower responsiveness to various mitogens such as Con A, PHA, PWM, and LPS than those of saline-treated mice. Low responsiveness to alloantigens was also observed. Flow cytometric analysis of the spleen cells revealed that Mac-1+ and Mac-2+ cells had increased while T and B cells had decreased in the BCG-vaccinated mice compared with the saline-treated mice at the time when the maximum level of inhibition of mitogen responses of BCG-vaccinated mice was observed. This suggests that the decreased in vitro immune response was due to the increase in macrophages which suppress lymphocyte functions. Support for this interpretation comes from the following two findings: (1) the restoration of mitogen responses of spleen cells when macrophages were eliminated by plastic adhesion or FACS sorting and (2) resuppression of PHA and Con A responses of plastic-nonadherent spleen cells by addition of adherent cells or flow cytometrically sorted Mac-1+ cells obtained from BCG-vaccinated mice. These results indicate the generation of suppressor macrophages after BCG vaccination and suggest that these macrophages prevent the autoimmune pathogenesis leading to diabetes in NOD mice.     

Inhibition of normal allogeneic lymphocyte mitogenesis by a factor released from human tumor cells in culture

Summary Human tumor and normal cell lines in culture were examined for the release of factors capable of inhibiting lymphocyte blastogenesis. Supernatants from tumor cell cultures of melanoma, carcinoma (lung, colon, breast), sarcoma, and normal fibroblasts inhibited normal lymphocyte response to PHA. Only supernatants from the tumor cell lines C1 (colon carcinoma), 734B (breast carcinoma), and 231 (breast carcinoma) were found to inhibit both PHA and ConA responses significantly. The two breast carcinoma cell lines, 734B and 231, which also were capable of inhibiting lymphocyte responses to PPD and alloantigens, were investigated further. The inhibition of lymphocyte blastogenesis caused by the supernatant of these two cell lines could not be overcome by the addition of added mitogen. Further experiments showed that the inhibition was not due to nutrient deficiencies and the supernatants were not directly toxic to the lymphocyte cultures as judged by trypan blue exclusion.     

Application of Concanavalin A during immune responsiveness skin‐swelling tests facilitates measurement interpretation in mammalian ecology

The skin‐swelling test is a simple and widespread method used in field ecological research to estimate cellular immune responsiveness in animals. This immunoecological test is based on measuring the magnitude of tissue swelling response at specific times following subcutaneous application of an experimental pro‐inflammatory stimulant. In the vast majority of studies across vertebrate taxa, phytohemagglutinin (PHA) is used as a universal stimulant. Given the complexity of immune response activation pathways of PHA, however, interpretation of test results can be ambiguous. Goal of this study was to improve methodology of the skin‐swelling test to decrease this ambiguity. Here, we present an alternative protocol aimed at facilitating interpretation of skin‐swelling data for mammals. Based on previous evidence suggesting that mammalian T cells are readily activated by Concanavalin A (ConA) in vitro, we compared cellular immune responses in vivo to PHA and ConA as an alternative pro‐inflammatory stimulant in mice. We measured magnitude of tissue swelling and compared it with intensity of blood cell infiltration into tissue over a 72‐hour interval. Our results corroborate that PHA and ConA show important differences in both dynamics and response amplitude in rodents. ConA induces stronger swelling with a distinct leukocyte activity pattern and higher pro‐inflammatory cytokine (interleukin 6 [IL‐6] and interferon gamma[IFN‐γ]) expression than PHA during peak response (24‐h post‐treatment). Furthermore, unlike PHA, magnitude of swelling was positively associated with cellular activity (number of neutrophils infiltrating tissue) following ConA injection. We conclude that ConA is the more suitable stimulant for skin‐swelling tests in mammals. This is because of the molecular binding specificity in the two lectins, that is, ConA specifically activates T cells while PHA also triggers erythroagglutination. We propose that ConA be used in all future ecological testing in mammals as it exhibits better performance and its application facilitates immunological interpretation of skin‐swelling test results.     

Immune responses in normal Indian langur monkeys (Presbytis entellus)--a primate model for visceral leishmaniasis

Abstract: The Indian langur monkey (Presbytis entellus) is an experimental host for a range of human diseases and for the assessment of vaccine candidate antigens to some common parasitic infections. This experimental host is particularly suitable for the follow‐up of immunological responses. To understand some of the mechanism that underlies the defense against experimental pathogens there is a need of the basic knowledge on antibody and cell mediated immune responses. In the present study 25 naïve monkeys were subjected to for assessment of their antibody responses to various human parasitic antigens as well as mitogen induced cellular responses. Only few monkeys were found to have low titer of antiparasitic antibodies. There was compressive dose dependent proliferative response of peripheral blood mononuclear cells. Unlike humans, the blastogenic as well as cytokine responses (IFN‐γ, IL‐2 and IL‐4) to Con A was considerably higher as compared to PHA. These findings are similar to what have been reported in other non‐human primates, confirming the appropriateness of Indian langurs for pre‐clinical trials.     

Ontogeny of con A and PHA responses of chicken blood cells in MHC-compatible lines 6(3) and 7(2)

The development of T cell responsiveness to Con A and PHA was examined in two MHC-compatible inbred chicken lines, RPRL 6(3) and 7(2), at ages 2 to 118 days posthatching. These lines are respectively resistant or susceptible to Marek's disease, a naturally occurring, virally induced T cell lymphoma. Between-line comparisons were made of optimal in vitro responses of diluted serum-free blood cells to each mitogen in two groups of chicks tested over ages 2 to 63 and 41 to 118 days. Over 2 to 63 days, Con A responses increased with age at the same rate in each line, but 7(2) responses averaged 2.3 times higher than 6(3). The increase with age was dependent on blood lymphocyte counts, which also increased with age in parallel in both lines. In contrast, the between-line difference in responsiveness was dependent on intrinsic reactivity of cells as well as lymphocyte counts. Covariance analysis was used to estimate that line 7(2) was 1.4 times higher than 6(3) in intrinsic cell reactivity, after accounting for the effect of the twofold higher blood lymphocyte counts in 7(2), and that this intrinsic difference contributed almost one-half the total difference. Over 41 to 118 days Con A responses no longer increased with age, although lymphocyte counts were still increasing, and the line difference (2.6 times) was now almost entirely contributed by a 2.3-fold superiority of 7(2) blood cells in intrinsic reactivity. The line difference in PHA responses was the reverse of the above in young chicks, with 6(3) responses greater than 7(2) in spite of lower lymphocyte counts. In additional chicks tested over 5 to 26 days, intrinsic reactivity of 6(3) cells to PHA averaged 4.5 times higher than 7(2). There was an abrupt decline in intrinsic reactivity of line 6(3) blood cells between 26 and 41 days to a level equal with 7(2). After this age, line 7(2) responses were 1.8 times greater than those of 6(3), and this difference was dependent solely on lymphocyte count differences. The results suggest that different gene systems mediate blood cell responses to PHA as compared with Con A. The pattern of developmental differences between inbred lines indicates the existence of distinct or partly overlapping T cell subsets with different reactivities to PHA or Con A, and of higher suppressor activity of adherent cells in line 6(3) blood. Both these differences may be related to line 6(3) inherited resistance to Marek's disease.     

Membrane characteristics of old and Rauscher leukemia virus infected mouse red blood cells

Some membrane characteristics of normal and Rauscher leukemia virus (RLV)-infected mouse red blood cells (RBC) were compared, both with regard to total populations and young and old groups of cells. Osmotic fragility, density distribution of cells and agglutinability by poly- -lysine (pLys), concanavalin A (ConA), phytohemagglutinin (PHA) and soybean agglutinin (SBA), were examined. RBC from RLV-infected mice were agglutinated at a higher rate and to a higher degree than normal mice RBC by pLys and by the lectins PHA and ConA. These RBC were generally osmotically more resistant and contained a young cell population of unusually high specific gravity. Comparison of RBC from RLV-infected mice with old RBC from normal mice showed some common membrane characteristics. Similarly to old RBC, RBC from RLV-infected mice have a high specific gravity and high agglutinability by pLys. However, they differ in that the RBC from RLV-infected mice are osmotically more resistant and are agglutinated by ConA; they are also agglutinated at a higher rate by PHA.     

Concanavalin A induced apoptosis in murine macrophage PU5-1.8 cells through clustering of mitochondria and release of cytochrome c

Concanavalin A (ConA), normally a mitogen of T-lymphocytes, was found to be a cell cycle-independent apoptosis-inducing agent in cultured murine macrophage PU5-1.8 cells. This assertion is based on the following observations: (1) ConA increased the number of cells with hypo-diploid DNA in a dose dependent manner as revealed by flow cytometry; (2) ConA elicited DNA fragmentation and the cytotoxicity of ConA was suppressed by -D-methylmannoside which blocks the lectin site of ConA; (3) ConA was able to release cytochrome c (cyto c) into the cytosol of PU5-1.8 cells. When isolated mitochondria were incubated with ConA, release of cyto c was observed too. Interestingly, clustering of mitochondria was found in the cytosol under a confocal microscope after ConA treatment. When cells were incubated with ConA-FITC and subsequently with mitotracker red (a probe for mitochondria), co-localization of fluorescence signals was observed. These results suggest that ConA was delivered to the mitochondria, induced mitochondrial clustering and released cyto c. Our results also show that introduction of exogenous cyto c electroporationally into ConA-untreated cells elicited DNA fragmentation. On the other hand, introduction of specific antibody against cyto c into PU5-1.8 cells suppressed the ConA-mediated cell death. Taken together, our results indicate that ConA induced apoptosis in PU5-1.8 cells through mitochondrial clustering and release of cyto c and the release of cyto c was sufficient to elicit apoptosis in PU5-1.8 cells.